The proposition that an oligonucleotide could be used as a therapeutic agent was described in 1978 by Zamecnik and Stephenson in a study on the growth inhibition of Rous sarcoma virus [Zamecnik, Stephenson; 1978]. Since that time, there has been a great interest in unmodified and modified oligonucleotides. As a result, new synthetic methods to selectively design oligonucleotides are important. Therapeutic oligonucleotides which interact specifically with a target mRNA by Watson-Crick base pairing are known as antisense oligonucleotides. Therapeutic oligonucleotides which interact with genomic DNA by means of Hoogsteen base pairing in the major groove, forming a triple helix, are known as triplex oligonucleotides.
In order for an oligonucleotide to be an effective therapeutic agent, the following issues must be considered: (1) cellular uptake; (2) degradation (by intracellular exo- and endonucleases); (3) triplex stability; and (4) triplex specificity [Geisow, 1991; Henene, 1991; Stein, 1993]. Most of these issues are addressed by modifying the various components of an oligonucleotide [Uhlmann, 1990]. For example, one important modification is the phosphorylation of the 3' and 5' end of an oligonucleotide to produce a 3',5'-diphosphate oligonucleotide. A 3',5'-diphosphate oligonucleotide not only provides protection against intracellular degradation, it also allows for the easy attachment of useful functional groups. These attached functional groups may be used to increase the effectiveness in therapy.
Currently, there are methods to produce oligonucleotides phosphorylated at the C-3' terminal [Gough, 1983; Volkov, 1988; Felder, 1984]--and methods to produce oligonucleotides phosphorylated at the C-5' terminal [Uhlmann, 1986]--however, there are no simple methods available for the preparation of an oligonucleotide phosphorylated on both ends of the chain. In addition, most of the phosphorylation methods are incompatible with phosphoroamidite chemistry, and hence incompatible with most automatic DNA synthesizers. For example, one successful phosphorylation method is achieved by enzymatic means with polynucleotide kinase and ATP [Maniatis, 1982]. Thus, improved phosphorylation methods are needed--especially those that can work on an automatic DNA synthesizer.
This invention demonstrates a simple method for the preparation of oligonucleotides phosphorylated on both ends of the chain (i.e.: 3',5'-diphosphate oligonucleotides). In addition, this method is well suited for use in an automatic DNA synthesizer.